Mechanisms underiying the complex interrelationships of infecfion and graft survival during induction and maintenance of transplantation tolerance are not well understood. Our goal is to understand how infection blocks the induction of peripheral and central tolerance. We have developed several innovafive technologies for identifying virus-immune T cells and determining their anfi-viral and cross-reacfive alloreacfivity at the single cell level. We have also developed methods for 1) quantifying alloreactive T cells using a 'synchimera' model based on CD8+ TCR Tg mice, 2) identifying naive and effector alloreactive T cells by their rapid production of cytokines following alloanfigen sfimulation, and 3) quanfifying in vivo CDS T cell effector function using an in vivo cytotoxicity assay. We will use these techniques with an exciting new technology for in vivo delivery of siRNA to block of CD40-CD154 interaction. These new technologies will allow us to test our overall hypothesis that inducfion of pro-inflammatory cytokines and IFNI is a fundamental mechanism by which innate immune acfivafion modulates the inducfion of peripheral and central tolerance. Specific Aim 1 is to determine mechanisms by which TLR ligation or virus infection modulates the induction of peripheral tolerance. We will test the hypothesis that innate immune activation by TLR agonists or virus infection abrogates the induction of peripheral tolerance through the producfion of pro-inflammatory cytokines and IFNI. Specific Aim 2 is to determine mechanisms by which TLR ligafion or virus infecfion modulates establishment of hematopoietic chimerism and central tolerance. We will test the hypothesis that the induction of peripheral and central tolerance involves mulfiple different but overiapping mechanisms. This project should reveal the mechanism(s) by which infection compromises the induction of peripheral and central transplantation tolerance. This Project will interact closely with Project 2 studying the maintenance of tolerance, and Project 3 studying how alloreactive CDS T cells die by apoptosis following cosfimulation blockade. These discoveries will be translated to human immune systems in Project 4 using both the Viral and Technology Core and Animal Core as critical resources for the accomplishment of our research goals.